The invention relates generally to the genetic transformation of plants and, more particularly, to a method for producing a transgenic monocotyledonous plant by introducing foreign genetic material directly into a node segment of a non-transgenic plant and then regenerating and selecting for a transgenic plant. Publications referred to in this specification are alphabetically listed at the end of the description.
The agriculture industry has benefitted from recent advances in the field of biotechnology that have enabled the development of hardier varieties of plants for crop improvement and ornamental purposes. In particular, the use of sophisticated recombinant DNA techniques has resulted in the production of genetically improved monocotyledonous grass cultivars, including cereal crops, forage grasses and turfgrasses, and dicotyledonous cultivars, such as tobacco, having increased yields, herbicide resistance, disease resistance, pest resistance, stress tolerance, durability, and the like.
The importance of improved cereal crops (e.g. maize, wheat, rice, barley) and forage grasses, such as ryegrasses (Lolium ssp.), orchardgrasses (Dactylis ssp.) and fescues (Festuca ssp.), is well recognized. Turfgrasses are important for providing attractive ground cover, such as for residential lawns, and for providing a support for many recreational sports, such as baseball, soccer and golf. The maintenance of golf courses, for example, requires intensive turfgrass management programs, including pesticide and herbicide treatments to prevent diseases and remove unwanted weed grasses. Therefore, production of turfgrasses that are herbicide-resistant and disease-resistant is beneficial to the turfgrass industry. The turfgrass seed market is second only to that of hybrid seed corn in being the largest seed market in the US.
Monocotyledons are generally recalcitrant to manipulation in vitro and to genetic transformation. Although transfer of foreign genes into plants by infection with Agrobacterium tumefaciens containing plasmid DNA is routine for many dicotyledons that readily form callus after wounding, the procedure is not routinely applicable in monocotyledons in which callus formation and transformation is more difficult. In addition to Agrobacterium, two other conventional methods of gene delivery, protoplast transformation and biolistic transformation, have been used to produce transgenic monocotyledonous and dicotyledonous plants.
Until the present invention, regenerable embryogenic tissue cultures have provided the major resource for the genetic transformation of monocotyledons. For example, mature seeds have been commonly used to initiate embryogenic callus cultures to establish suspension cell cultures of cereals, forage grasses and turfgrasses. However, genotypic variation in outbreeding species, such as common ryegrasses, has been problematic because each seed-derived embryo possesses a unique genotype and it has been shown that standard culture conditions to induce embryogenesis are not optimal for every genotype. Therefore, embryogenic and suspension cell cultures from various sources have had to be selectively established on a species to species and genotype to genotype basis. Immature embryos and inflorescences have been used for callus initiation with pearl millet (Pennisetum americanum), Guinea Grass (Panicum maximum Jacq.), Napier Grass (Pennisetum purpureum Schum.) and perennial ryegrass (Lolium perenne). Basal sections of leaves have been used for callus initiation of orchardgrass (Dactylis glomerata L.) and tall fescue (Festuca arundinacea Schreb.). Chopped mature embryos have been used directly to establish suspension cultures of tall fescue, perennial ryegrass and Italian ryegrass (Lolium multiflorum); and both immature caryopses and mature seeds have been used to obtain embryogenic suspension cultures of Italian ryegrass and tall fescue (Lee, 1996). Regeneration of fertile plants has been obtained from isolated vegetative meristems of Italian ryegrass and perennial ryegrass (Perez-Vicente et al., 1993), from shoot apex explants from foxtail millet (Setaria italica L.), and from suspension culture-derived protoplasts of meadow fescue (Festuca pratensis Huds.). Switchgrass (Panicum virgatum L.) plants have been micropropagated from axillary buds in node segments in tissue culture, but there are no reports of transformation (Alexandrova et al., 1993).
Stable transformed callus lines and transgenic plants from callus of perennial ryegrass have been established. Transformed callus lines of Italian ryegrass have also been reported but transgenic plants have not been regenerable from these callus lines. Kentucky bluegrass (Poa pratensis L.) plants can be regenerated from protoplasts, embryogenic suspension cultures and seed-derived callus cultures, but there are no reports of transformation. Until the present invention, there have also been no reports of transformation of St. Augustinegrass (Stenotaphrum secundatum Walt! Kuntze), although plants have been regenerated from immature embryo-derived callus (Kuo et al., 1993).
Protoplast transformation has been used to obtain transgenic turfgrasses, including orchardgrass, tall fescue, creeping bentgrass, redtop and red fescue. Direct DNA uptake by the protoplasts was enhanced by the use of polyethylene glycol (PEG) or electroporation. However, protoplast transformation has only been successful in a few cultivars of cereal crops and regeneration of cereal plants from protoplasts appears to be strongly genotype and culture condition dependent. Moreover, for regeneration of turfgrass protoplasts, nurse cells in the protoplast cultures are required. These have reportedly been provided by addition to the culture of agarose beads with adhered cultured nurse cells or by a nurse cell feeder layer (Lee, 1996).
Microprojectile (biolistic) bombardment has been used successfully to produce transgenic turfgrass, such as creeping bentgrass (Hartman, et al., 1994), perennial ryegrass, tall fescue and red fescue, from embryogenic callus and suspension cultures. Biolistic bombardment employs high velocity metal particles to deliver biologically active DNA into plant cells. The concept was first described by Klein et al. (1987) and has become a successful DNA delivery method in a number of plants. An advantage of biolistic transformation is that a variety of recipient cell types can be used, including embryogenic callus, suspension cell cultures, immature or even mature embryos. For those monocotyledonous species and cultivars that form embryogenic cultures, but a protoplast regeneration system is not easy to establish, biolistic genetic transfer is a possible alternative method for cell transformation.
Successful genetic transformation of dicotyledonous plants, particularly tobacco plants, have been reported. Transgenic callus cell lines of tobacco have been recovered after microprojectile bombardment of tobacco leaves (Tomes, et al.) and suspension cell cultures (Klein et al., 1988) and transgenic tobacco plants were regenerated after biolistic bombardment of tobacco leaves. There have also been reports of successful transformation of chloroplasts of tobacco using the biolistic process.
Transformation of dicotyledonous sunflower and soybean cells from a cotyledonary node associated with an apical meristem has been reported. However, successful transformation required tissue culture of the node cells in the presence of a cytokinin, prior to transformation, to induce the cells to differentiate into meristematic tissue cells and to become synchronized in the cell cycle (Tomes et al., 1991).
Transgenic perennial fruit plants (dicotyledons), such as cranberry, have been obtained from transformed stem tissue after biolistic bombardment. However, successful transformation of the stem tissue required prolonged tissue culture in the presence of a cytokinin, prior to bombardment, to induce the formation of adventitious buds (Serres et al., 1993).
Transformation efficiencies (the ratio of the number of cells transformed to the number of starting cells) are low, however, for each of the above-described transformation methods, being on the order of 10.sup.-3 to 10.sup.-6.
In addition to the commonly used transformation techniques, available alternative methods for the direct introduction of foreign DNA into cells include silicon carbide fibers, electroporation of intact tissues, electrophoresis and microinjection. However, the stable expression of foreign genes, using these techniques, has yet to be established.
In view of the foregoing uncertainties and, in many cases, unreliability of current methods for successful transformation and regeneration of transgenic plants, there is a need for an efficient method of genetic transformation and regeneration that can be successfully applied to monocotyledonous plants, and particularly to turfgrasses, to obtain transgenic plants. Moreover, there is a need for a method of transforming monocotyledon plant cells that does not require extensive cell culture preparation and/or callus formation and/or extended tissue culture. Further, there is a need for improved techniques for genetic transformation and plant regeneration and selection systems for monocotyledons that allow introduction of foreign genetic material that alters the physical, biochemical and/or physiological properties of the plant to provide beneficial traits or reduce unwanted characteristics.